Medical diagnosis and treatment of diseases at the genetic level is quickly becoming a reality. Drug design strategies will increasingly depend on developing new methods for regulating gene expression. Early detection of infectious viral diseases and genetic mutations using fast, reliable diagnostic techniques combined with gene-therapy strategies create the possibility of effecting a cure before symptoms of the disease appear. New technologies based on gene isolation and purification, synthesis, amplification, and detection are required to meet these challenges. These emerging technologies require improved methods for oligonucleotide immobilization in several key fields including: nucleic acid separation/purification; nucleic acid amplification (solid-phase PCR); oligonucleotide synthesis; isolation of nucleic acid binding proteins and drugs; detection of oligonucleotides through hybridization; and sequencing.
Amplification of Oligonucleotides by Solid Phase PCR
The polymerase chain reaction (PCR) is a rapid procedure for producing many copies of a specific segment of DNA in vitro. This technique has now made possible many applications such as molecular genetic research, gene sequencing, forensic/criminal and clinical investigations and many others in which only a minuscule quantity of DNA is available. PCR was originally developed for the solution phase and requires four essential ingredients; Taq DNA polymerase which is the enzyme responsible for building the new DNA copies, the original DNA strand to be amplified, the four triphosphate bases, and finally, the priming sequences from which the new DNA copies will grow. A much improved method is to attach the priming sequences to a solid support which allows the amplified DNA to be chemoselectively removed after the reaction is completed.
Oligonucleotide Synthesis Via Solid Phase Methods
Short fragments of single-stranded DNA or RNA of any desired sequence can be rapidly synthesized using an automated DNA synthesizer which is now commonplace in many laboratories. Oligonucleotides are made by the sequential addition of activated monomers to a growing chain that is linked to an insoluble solid support. The solid-phase synthetic method has the following advantages: reaction yields can be near-quantitative by using excess reagents which can then be easily removed by filtration processes; the repetitive synthesis is readily automated; handling is minimized thereby decreasing the risk of contamination; and wastage of expensive reagents is also minimized. Functionalized polystyrene beads or carboxyl-derivatized controlled-pore-glass are commonly used as supports, with the powdered material being sealed into a tube which has porous frits at both ends. Usually, the first nucleotide is attached to the solid support via a carboxylic ester link to the 3' hydroxyl, and synthesis is carried out in the 3'-5' direction. A high ratio of oligonucleotide to surface area is required to optimally perform the synthesis to prevent wasteage of reagents. It is advantageous to attach special tethering groups to one of the chain ends to act as a chemical "handle" so that the single-stranded nucleic acid can be attached to other solid surfaces such as affinity columns or biosensor devices. Solid phase synthesis and the immobilization technology to which it is dependent, can be applied to the synthesis of other types of biopolymers including peptides, proteins, and combinatorial synthesis of diverse arrays of any class of molecule.
Separation, Isolation and Purification of Oligonucleic Acids, Oligonucleotide-Other Molecule Complexes and Other Biomolecules
Biomolecules in general can be purified by electrophoretic, chromatographic, filtration or by affinity techniques. Electrophoresis is widely used to separate nucleic acid fragments in a gel matrix. The fragments are usually transferred or "blotted" onto a membrane which has an affinity for the nucleic acids so that further processing can be accomplished. Reverse-Phase Liquid Chromatography (RPLC) has been used to separate mixtures of nucleic acids, proteins and other biomolecules on coated solid supports. Microfiltration is used to remove impurities from biomolecule preparations.
Improved separations can be achieved by immobilizing various sequences of nucleic acids onto the stationary media to produce an "affinity" hybrid technique. Single-stranded nucleic acids can be immobilized onto a solid support, to which the complementary strand can specifically hybridize; such a technique is referred to as hybridization. Impurities are washed away, while the complementary strand remains affixed, and elution selectively occurs when variables such as buffer strength are changed. In such a manner, improved electrophoresis membranes for "Northern blots", nucleic acid chromatographic supports, and nucleic acid binding filtration media can be made.
Molecules other than nucleic acids can specifically recognize immobilized nucleic acids. Research to discover new treatments for genetic diseases requires the development of novel methods for investigating the interactions of genes with regulatory proteins. Gene transcription, replication and repair are mediated by many DNA or RNA binding proteins. Drugs such as cis-diaminedichloroplatinum (II) known as "cisplatin" which has antitumor activity for the treatment of ovarian, bladder and testicular cancer, anthracycline antibiotics and polycyclic aromatic compounds can intercalate into DNA structures. Antisense drug therapy innovations are directed at strongly binding a complementary segment of nucleic acid material to the target gene in a highly selective fashion. Similar to nucleic acid purification through immobilized hybridization as mentioned earlier, proteins, drugs and any type of nucleic acid binding molecule can be purified through selective interactions with immobilized oligonucleotides. In all cases, a high density of immobilized oligonucleotides will result in increased efficiency, together with minimization of waste production.
Detection of Oligonucleotides, Antisense Compounds and Small Molecules
Detection of oligonucleotides for diagnostic assays through hybridization and sequencing is also dependant on high density surface immobilization of oligonucleotides. Determining genetic sequences is a well established field. Standard techniques such as electrophoretic separation of partially digested nucleic acid fragments are generally too slow for clinical work. A different approach is sequencing by hybridization or SBH in which a library of short oligonucleotide probes, labelled in some way, and of known sequence, are presented to unknown DNA. When complementary sequences are found, a process known as hybridization occurs which allows for signalling the presence of a particular sequence in the gene. New techniques such as micromachined capillary electrophoresis arrays require high density immobilization techniques, as each channel possesses a very minute surface area.
Immobilized nucleic acid probes on sensor surfaces can provide much faster analyses at a fraction of the cost. Such is the basis for a "gene chip" in which vast arrays of different genetic probes, approximately 10-30 bases in length are immobilized onto a silicon wafer similar to those used in computer chip manufacture. The parallel revolution in microelectronics, combined with advances in automated nucleic acid synthesis has generated the development of new biosensor devices for the analysis of gene sequences and drug discovery schemes. A biosensor is a device which transforms biological information into electronic form which can then be readily interfaced with computer technology. Most biosensors include a biochemical sensor element structure comprising a platform, usually a solid surface, to which the biologically active probe molecules are attached. Biomolecules such as DNA are extremely selective, and can efficiently bind to a specific target molecule in a solution containing many other species. The probe-target interaction is then observed by a mechanism (optical, electrochemical or piezoelectric) which can transduce chemical information to electronic data. All of these techniques can deliver information in real-time, which is a benefit that the standard techniques do not possess.
The nucleic acid probe should be immobilized to the surface in some way so that the biosensor can be continuously reused in a flow injection analysis (FIA) format. The nucleic acid probes must be immobilized in the correct concentration, under mild conditions, rapidly, among many other considerations. The biosensor surface can be tailored so that more than one type of nucleic acid is attached to its surface. The use of photoprotective schemes has been reported as being capable of producing patterned surfaces.
Immobilization Methodology
Numerous techniques have been developed for the immobilization of enzymes and antibodies (Mosbach (ed.), Methods in Enzymology, Vol. 137, 1988) and many of the techniques used to immobilize proteins can also be adapted for nucleic acids (Dunlap, Advances in Experimental Medicine and Biology). Adsorption is the simplest method to attach nucleic acids to surfaces, since no reagents or special nucleic modifications are required. Non-covalent forces affix the nucleic acid to such materials as nitrocellulose, nylon membranes (Brent et. al., Current Protocols in Molecular Biology, 1993) polystyrene or metal oxide surfaces such as palladium or aluminum oxide. The main disadvantages of these methods are that the nucleic acid may be readily desorbed from the substrate by hybridization conditions, and the base moieties may be unavailable for hybridization if they are bonded to the substrate. Crosslinking or entrapment (Licache et. al., Nucleic Acids Res., 1994, 22, 2915) in polymeric films has been used to create a more permanent nucleic acid surface. The nucleic acid can be crosslinked by exposure to U.V. light (pyrimidine-pyrimidine dimer). Vinyl-substituted nucleotides have been made which can polymerize (Pitha, Polymer, 1977, 18, 425). The nucleic acid can be embedded in an amino-containing dextran matrix (Johnsson et. al., Anal. Biochem., 1991, 198, 268) or aminoethylcellulose crosslinked with gluteraldehyde, silica, or in polyacrylamide.
Avidin/streptavidin-biotin complexation has found considerable application in the nucleic acid biosensor field (Ebersole et. al., J. of the Amer. Chem .Soc., 1990, 112, 3239). Avidin and streptavidin are large proteins (70 kD) which each contain 4 biotin binding sites. Biotin is a small molecule which attaches with very high affinity to the binding site (K.sub.d =10.sup.-15 M), and can only be removed under the most extreme conditions. The avidin is first adsorbed onto the substrate, and is then exposed to an aqueous solution of biotinylated nucleic acid. The inherent aqueous stability of avidin and biotin makes the system easy to handle. However, the presence of the large protein layer may present non-specific binding sites and compromise the sensitivity and selectivity of certain types of sensors.
Alternatively, the nucleic acid can be constructed with a thiol linker which can be used to directly complex to gold surfaces (Ito, et. al., Anal. Chim. Acta., 1996, 327, 29). It is desirable to fashion assemblies similar to the long-chain self-assembled monolayers of alkanethiols which have been described in the literature (Van Ness et. al., Nucleic Acids Res., 1991, 19, 3345). The thiol-nucleic acid probably cannot produce a close-packed surface due to the large hydrophilic nucleic acid group, and therefore its stability is questionable.
It is desirable to attach the nucleic acid covalently to a surface of the support by a linker attached to one of the ends of the nucleic acid chain. By doing so, the nucleic acid probe is free to change its conformation so that hybridization can take place, yet cannot be displaced from the sensor. Much work has centered in this area, with early attempts being based on attaching the 3' hydroxyl or phosphate group to carboxyl residues on various celluloses using carbodiimide derivatives (Schott, Affinity Chromatography: Template Chromatography of Nucleic Acids and Proteins, 1984). Cyanuric chloride (Biagione et. al., Anal. Biochem., 1978, 89, 616) has been used to react oligonucleotides to a variety of materials. Cyanogen bromide (Scowten, Affinity Chromatography: Bioselective Adsorption of Inert Matrices, 1986) has been used to link one or more exocyclic amine residues to agarose via isourea ether groups. Carboxylic acid and aldehyde modified nucleic acids have been attached to latex spheres via hydrazide or Schiff-base type linkages (Kremsky et. al., Nucleic Acids Res., 1987, 15, 2891).
Many biosensor surfaces consist of silica or metal oxide. Such surfaces need to be first modified with some type of adhesion agent (EP Application No. 96-303245). Organosilanes such as aminopropyltriethoxysilane (APTES) (Wu, et. Al., Chinese J. Microbiol. Immunol., 1990, 23, 147). 3-mercaptopropyltriethoxysilane (MPS) (Bhatia et. Al., Anal. Biochem. 1989, 178, 408) and glycidoxypropyltriethoxysilane (GOPS) (Maskos et. Al., Nucleic Acids Res., 1992, 20, 1679) have been used to created functionalized surfaces on glasses, silicas, optical fibers, silicon, and metal electrodes to name a few. The silanes hydrolyse onto the surface to form a robust siloxane bond with surface silanols, and also cross-link to further increase adhesion. In the case of APTES, succinnic anhydride is often used to change the amino functionality to carboxylic acid which is then attached to an amino-linked nucleic acid via carbodiimide coupling. MPS can be used to form disulfide linkages with thiol-containing biomolecules. GOPS has been used in schemes using long polyether chains to provide greater distance and flexibility between the surface and the nucleic acid probe.
Alkyl silanes have been extensively used to immobilize a wide variety of biomolecules to surfaces. The alkoxy or chloro leaving groups are particularly reactive towards hydroxyl groups found on glass, quartz, silicon and metal oxide surfaces. The surface hydroxyl group attacks the silicon in an Sn2 reaction, and the new Si.sub.(surface) --O--Si.sub.(silane) bond is a siloxane-bond. Monoalkoxy or monochloro silanes can only form one siloxane bond to the surface, and therefore, the degree of surface coverage by the silane is limited by the number of available surface hydroxyl groups, which in the best of cases (glass) is no more than about 4 hydroxyls per nm.sup.2. Di or tri alkoxy or chloro silanes are capable of forming more than one siloxane bond. The quantity of surface hydroxyl groups per unit area is generally too low for the silane to form more than one siloxane bond to the surface. Instead, the silanes can crosslink together to form two dimensional or multilayer networks on the surface, and therefore bridge the gap between surface hydroxyls and increase the degree of surface coverage.
This intersilane crosslinking requires a sufficient water to be present, e.g. a stoichiometric quantity of water, for the polymerization to occur, and can be carried out in the solvent used for the silanization reaction, or can be supplied by water which is adsorbed to the substrate surface. The actual mechanism of silanization depends on the conditions used. In solution, the commonly accepted mechanism is a three-step process, the first step being the hydrolysis of the chloro moieties of a silane such as octadecyltrichlorosilane (OTS) at the hydroxylic substrate surface to generate a silanetriol, which then physisorbs onto the substrate via hydrogen bonding and ultimately forms both Si.sub.substrate --O--Si.sub.silane and Si.sub.silane --O--Si.sub.silane cross-linking type of covalent bonds (Sagiv, J. of the Amer. Chem. Soc., 1980, 102, 92). However, it has been shown that hydrolysis of the chloro entities of OTS occurs in the bulk solution phase instead of at the substrate surface as envisaged earlier (Angst, et. al., Langmuir, 1991, 7, 2236).
The degree of surface coverage depends on several variables such as reaction time, temperature, degree of hydration of the substrates, nature of the solvent, the cleaning procedure utilized prior to silanization of substrates and the nature/morphology of the oxide layer on the substrate. Silberzan et al. (Langmuir, 1991, 7, 1647) as well as Angst and Simmons (Angst, et. al., Langmuir, 1991, 7, 2236) obtained a tightly-packed monolayer of OTS on a fully hydrated oxidized silicon wafer surface, while with a dry silicon wafer a lower surface coverage resulted. Tripp and Hair (Langmuir, 1992, 8, 1120), through an IR spectroscopic study, showed that no direct reaction occurs between OTS and either the silica surface hydroxyl groups or even the first water layer bound to the fumed silica surface. Despite the growing body of evidence concerning the importance of surface-moisture, there is not yet a standard protocol that can be used to increase reproducibility of the silane films.
Alkoxysilane 3-mercaptopropyltrimethoxysilane (MPS) has been extensively used as an immobilization agent, however, there are several problems with this reagent. Caldwell (Yee et. al., Langmuir 1991, 7, 307) showed that a silver stained MPS surface appeared rough when examined by scanning electron microscopy (SEM), and the MPS surface consisted of submicrometer size particles. They acknowledged that the MPS silane produced a multilayered structure. The group of Sligar and Bohn (Hong, et. al., Langmuir, 1994, 10, 153) found that both a 17% MPS film (diluted with n-propyltrimethoxysilane) and a 100% MPS film have similar abilities to load cytochrome b.sub.5 at a 30% loading level. They performed a free-thiol assay using Ellman's reagent and discovered that the quantity of unreacted thiol groups after exposure to cytochrome b.sub.5 on the surfaces is nearly identical to the quantity of unreacted thiol groups before exposure to the cytochrome. The authors concluded that most of the cytochrome was non-specifically adsorbing to the MPS film, but did not hypothesize which functionality the protein was adsorbing to. In all likelihood, the cytochrome was physisorbing to the exposed silanol-containing backbone of the disordered MPS multilayer, or to exposed patches of glass not covered by MPS. They also acknowledged that MPS produces multilayer structures, and found that the masses of MPS can be hydrolytically removed from the surface.
Alkoxy silanes are predisposed to form disordered multilayered films. This effect is compounded when the alkoxysilane contains a short alkyl chain which reduces the silane's ability to self-assemble into highly ordered films. Under unsuitable conditions of the silanization process, the alkoxysilane will tend to polymerize in solution, possibly forming large aggregates, which then migrate to the substrate surface and then polymerize onto it. Other aggregates pile up on top of each other in an uneven manner until a film many times thicker than the length of one monomer builds up. Although this coating may still be usable for immobilization purposes, it is not efficient. Much of the functionalized end of the silane is not projected normal to the substrate towards the bulk solution, but instead is oriented in every conceivable direction including parallel against the substrate surface. Clearly, this represents a severe steric barrier, and that fraction of the surface is not available for nucleic acid immobilization, although small probes such as the silver ion may be able to penetrate inside the pores. The pores may trap potential interferant molecules which may complicate biosensor data interpretation.
Trichlorosilanes are much more reactive than trialkoxysilanes, and appear to form the densest films. If the alkyl group is from 8-18 carbons in length, the self-assembly process will cause the silanes to form a "monolayer"-like coating in which the alkyl chains are packed together to nearly the same density as crystalline polyethylene. The amount of surface area (Montgomery, et. al., Anal. Chem., 1992, 64, 1170) each alkyl chain occupies is about 20 .ANG..sup.2.
Bifunctional trichlorosilanes have been made so that other molecules can be later attached to the silanized surface. 1-Thioacetato-16-(trichlorosilyl)-hexadecane or related analogues have been described as a potential linking agent for biomolecules (Balachander, et. al., Langmuir, 1990, 6, 1621). In contrast, the short chain alkoxysilanes such as APTES, MPS, and GOPS which have been used to link nucleic acids to surfaces, usually consist of a 3-carbon tether, and tend to form disordered multilayer structures. It is possible to dilute the active silane monomer with a monomer which does not contain the linking group. For example, a simple methyl-terminated "diluent" monomer could be used to effectively "space-out" the active silane monomers that deposit on the surface.
There are many benefits that can be gained by using trichlorosilane linkers for biomolecule immobilization schemes. The avidin protein has a highly polar exterior, with many carboxylic acid and amine residues exposed. These could serve as potential binding sites for the nucleic acid probe or target molecules, or could adsorb contaminating materials such as other proteins from the solution, all of which could be detrimental to the operation of the sensor. Methyl terminated diluent silanes provide a hydrophobic alternative to polar materials which may attract unwanted contaminants. Other diluents could be used to provide other functionalities to the surface, for example, the diluent could contain alcohol groups to increase hydrophilicity, and the surface properties could be readily controlled. The number of carbons in the diluent could also be varied to control the steric environment around the active silane's functional moiety. Most importantly, the active silane could be synthesized to have a wide variety of functional groups for immobilization.
The tether group is required to supply the oligonucleotide with a reactive functionality so that it can be chemically manipulated, and to allow the oligonucleotide to extend any specified distance away from the surface (French Patent Application No. 94-12972). Thiol-tethered oligonucleotides have been immobilized onto bromoacetyl-derivatized polyacrylamide supports (U.S. Pat. No. 5,478,893).
It is desirable to be able to create both permanent and reversible linkages between the nucleic acid and the surface. One patent (U.K. Patent Application No. 89-21605) describes a phosphorus-sulfur bond placed in the backbone of an immobilized oligonucleotide which was cleaved by silver nitrate. Sulfur can also be used in a completely different way in the form of the thiol group, which can form two main types of linkages: disulfide and thioether. The reversible disulfide bond can be created using the specific reaction known as thiol-disulfide interchange, in which a thiol containing molecule reacts with a disulfide-containing molecule, so that one of the ligands from the disulfide is transferred to the original thiol group to form a new disulfide. The disulfide can be part of a bifunctional coupling agent (U.S. Pat. No. 5,399,501). The disulfide can be cleaved specifically under very mild conditions with a variety of reagents such as dithiothreitol (DTT) for example, which will regenerate the free thiol. A permanent thioether bond can be created from a thiol and a variety of reagents which contain reactive leaving groups. Thiol surfaces have been used for covalently bonding biologically active compounds (U.S. Pat. No. 4,886,755). Halobenzylic compounds readily undergo reaction with thiols, and the resulting thioether bond is very resistant to cleavage.
Solid supports carrying nucleic acids, enzymes, peptides and other biomolecules for the purposes set out herein should have a high surface density of attached groups. This is desirable so that the device can operate at maximum sensitivity in detecting and binding affinity biomolecules to be selectively bound and removed for analysis. It is also desirable to allow devices of small physical size to be prepared, for analysis of very small quantities of test reagents.
It is an object of the present invention to provide novel biochemical sensor element structures which have a very high surface density of biochemical molecules attached to the surface of a solid support.
It is a further object to provide a processes for preparation of such element structures.